Is it the undifferentiated stem cell, the endothelial progenitor cell, or the mature endothelial cell that has the greatest therapeutic potential for neovascularization? We hypothesize that it is the endothelial progenitor cell, and not the mature endothelial cell, that gives rise to more actively migrating, proliferating cells that are better able to build new vessels and adapt and integrate with the host microenvironment. The goal of the proposed research project is to investigate and compare the developmental stages at which maturing endothelial cells will optimally form new blood vessels and integrate with the host vasculature. Mouse embryonic stem cells (ESC) will be used for these studies because they provide an ideal in vitro system for stem cell differentiation. Cell populations will be isolated at 4 distinct stages of differentiation from an undifferentiated ESC through a mature endothelial cell. The vasculogenic and angiogenic potential of the 4 cell populations will be examined using in vitro collagen gel assays and the whole animal chick chorioallantioc membrane (CAM) assay. Specific aims are: Aim 1- Efficient generation and characterization of the 4 distinct stages of vascular differentiation. These will include: a.) undifferentiated ESC, b.) Flk-1 positive vascular progenitor cells, c.) outgrowths from Flk-1 positive vascular progenitor cells including EC and SMC, and d.) purified and expanded endothelial cells. Aim 2- Determine the precise developmental stage at which maturing endothelial cells will optimally generate new vessels in collagen gels. Aim 3- Determine the precise developmental stage at which maturing endothelial cells will optimally generate new vessels on the chick CAM. The generation of stem cells at the appropriate stage of development for the growth of new vessels will be important when using these cells for repair of dead heart tissue or damaged or diseased blood vessels.